1. Field of the Invention
This invention relates to aqueous gels useful for the separation of mixtures of components, especially mixtures of biochemical components such as nucleic acids and proteins, by electrophoresis.
2. Statement of Related Art
Aqueous gels based on agarose and certain derivatives of agarose are known to the art for use in the electrophoresis of substances of biochemical origin.
Nonderivatized (native) agarose products useful in gel form for electrophoresis are commercially available, e.g. as the SeaKem.sup..RTM. agarose line of products, a product of FMC Corporation, BioProducts Group, Rockland, Maine 04841, U.S.A.
A number of derivatized agaroses are disclosed in the prior art.
For example, U.S. Pat. Nos. 4,275,196 and 4,312,727 to Shainoff disclose gels containing glyoxal agarose for use in the electrophoretic separation of complex proteins. The glyoxal agarose is prepared by the reaction of agarose with glycidol to form glycerated agarose, which is then reacted with sodium periodate to form the glyoxal agarose. These patents, and/or the publication "Zonal Immobilization of Proteins and Peptides on Glyoxal Agarose" by J. R. Shainoff (1987) disclose using sodium orthoborate and/or cyanoborohydrides for the fixation or proteins in already formed handleable gels of glyoxal (not glyceryl) agarose.
"Affinity Chromatography", by W. H. Scouten, John Wiley & Sons, New York (pub.) (1981) discloses at section 3.1.1 that cyanogen bromide reacts with the vicinal diols of agarose to produce an "activated" agarose that subsequently can be coupled to spacer molecules containing primary amines. It is further disclosed that this method yields an (undesirable) bioselective adsorbent with anion-exchange properties.
U.S. Pat. No. 3,956,273 to Guiseley discloses alkylated, alkenylated, acylated and hydroxyalkylated agarose gels useful for electrophoresis.
U.S. Pat. No. 4,319,975 to Cook discloses a derivatized agarose gel useful for electrophoresis in which the molecular weight of the derivatizing substituent is between 100 and 1,000,000, and the substituent has a preselected conformational shape such that the pore diameter of the derivatized agarose is not reduced below 10 Angstrom units.
A number of derivatized agarose products are also currently on the market. Examples of these products are given below.
Hydroxyethylated agarose is a product of FMC Corporation, BioProducts Group, Rockland, Maine 04841 U.S.A. under the trademark SeaPlaque.sup..RTM..
NuSieve.sup..RTM. GTG.sup..RTM. agarose sieving gel is a product of FMC Corporation, BioProducts Group, Rockland, Maine 04841, U.S.A. Its use has been described in "Small DNA Fragment Separation and M13 Cloning Directly in Remelted NuSieve.sup..RTM. GTG Agarose Gels" by Dumais & Nochumson, BioTechnicues, 5:62 (1987). Buffer systems disclosed in FMC Corporation literature as commonly used with NuSieve GTG agarose include: TAE (40 mM Tris, 20 mM acetate, 2 mM EDTA at pH 8.0) and TBE (89 mM Tris, 89 mM borate, 2 mM EDTA at pH 8.0). NuSieve comprises a native agarose which has first been derivatized to hydroxyethyl agarose and has then been partially depolymerized.
Another known sieving agarose is a combination of one part SeaKem.sup..RTM. native agarose with three parts of the above described NuSieve.sup..RTM. agarose. This combination has been disclosed as useful in Polymerase Chain Reaction (PCR) procedures by Saiki, Gelfand, Stoffel, et al., in "Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase", Science. 239:486-491 (1988).
Still another known derivatized agarose sieving gel is SeaPrep.sup..RTM. hydroxyethyl agarose, also a product of FMC Corporation, BioProducts Group, Rockland, Maine 04841 U.S.A. There is a disclosure of the use of this product for sieving electrophoresis by Nochumson, S. in Electroohoresis '81, 213-218, Allen & Arnaud (eds.), W. de Gruyter & Co. (pub.), New York (1981).
Also, highly derivatized agarose in degraded form is a component of the ProSieve.sup..TM. gel system, a product of FMC Corporation, BioProducts Group, Rockland, Maine 04841 U.S.A. The ProSieve gel system forms discontinuous, thermo-reversible gels useful for the electrophoretic separation of proteins.
Other known sieving gel compositions include the mixture of agarose with hydroxyethyl cellulose as disclosed by Perlman, Chikarmane, and Halvorson in "Improved Resolution of DNA Fragments in Polysaccharide-Supplemented Agarose Gels", Analytical Biochemistry, 163:247-254 (1987).
Agarose (non-derivatized) is non-toxic, has high gel strength, low electroendosmosis and does not require free radical polymerization for gel formation. Agarose is a naturally occurring, substantially linear polysaccharide polymer which forms gels that are thermally reversible, thereby enabling separated components to be recovered from the melted gel.
Gels prepared with native (non-derivatized) agarose exhibit a characteristic coarse pore structure, a feature which renders them the preferred medium for the electrophoretic separation of large macromolecules. Generally speaking, primarily nucleic acids greater than 1000 base pairs can be resolved. Although smaller molecular weight entities can be resolved (restricted) by increasing the agarose content of the gel, this produces high viscosities in the agarose casting solutions, which make them very difficult to cast. Agarose gels are thus precluded from being used in a number of analytical and preparative procedures.
The large pore limitation of agarose gels can be diminished and their sieving action improved by forming the gels from certain agarose derivatives having a finer pore structure than the parent agarose. One preferred class of such modified agarose is hydroxyalkylated agarose produced by replacing 1 to 4 hydroxyl hydrogen atoms in the agarobiose units of the agarose polymer chain with hydroxyalkyl moieties. An especially preferred member is hydroxyethylated agarose obtained by reacting agarose with 2-chloroethanol in the presence of alkali. Gels from hydroxyethylated agarose are capable of resolving proteins of from about 50 kD to about 600 kD. Moreover, such gels have lower melting points than native agarose gels, an advantage when recovering sensitive biological substances from the remelted gels.
While derivatized agarose gels represent an advance in the art, both native agarose and the known derivatized agarose gels form casting solutions whose viscosity increases with gel concentration. This makes it difficult to prepare gels of sufficient concentration to achieve maximum sieving action.
Moreover, some of the above gels have elastic properties in need of improvement, i.e. the gels tend to break when handled because they are brittle. In addition, these prior art gels may provide inadequate separation of mixtures of proteins or relatively small DNA and RNA molecules unless the components of the mixture have relatively large differences in molecular weight.